The primary objective of the proposed research is to achieve a precise biochemical understanding of the cytoplasmic machinery involved in the selective transport of nascent proteins (precursors) into mitochondria. This machinery has been revealed by the detection of activities that appear to be proteinaceous extramitochondrial activities required for the import of proteins into mitochondria in vitro. No sustained effort to isolate and characterize the molecular species behind these activities has been reported in the years since their detection. The primary objective of this research is to isolate and characterize import factors from the cytosol of the yeast Saccharomyces cerevisiae. In the period covered by this proposal, the following aims will be pursued. 1) Published in vitro import systems will be adapted to produce assays useful for the purification of import factors. Progress towards this goal has already been made. 2) Crude extracts with import factor activity will be prepared from S. cerevisiae and the molecules responsible will be purified to homogeneity. 3) Purified factors will be characterized to determine the number of different factors, their substrate specificity, the nature of precursor/factor interactions, whether import factors act to alter the conformation of precursors, and the precise subcellular localization of the import factors. Purification of import factors is the necessary first step in the investigation of the cytoplasmic enzymology of protein import into mitochondria. It will set the stage for detailed studies on the biochemistry of factor action, for elucidation of the molecular biology of the cytoplasmic machinery of import, and finally a precise elucidation of the role of the cytoplasmic machinery in facilitating and possible regulating mitochondrial biogenesis. The understanding of a fundamental process such as mitochondrial biogenesis will be an important advance toward the goal of a complete molecular description of cellular physiology and pathology.